"Protein only" (prion) inheritance, operating at the level of protein conformations rather than nucleic acids, has bee found in such diverse organisms as mammals and fungi. Here, yeast is used to investigate the rules of prion inheritance. Factors influencing the appearance of yeast prions are emerging. The most intriguing is the prion-like element [PIN+], required for the de novo appearance of the [PSI+] prion. The gene encoding the [PIN+] protein, PIN1, will be identified from genetic screens. Disrupting and overexpressing PIN1 will test 2 hypotheses the non-prion form of Pin1 inhibits [PSI+] seeding, vs. the prion form of Pin1 catalyzes [PSI+] seeding. In vivo aggregation of Pin1 will be examined and simulated in vitro using purified Pin1. The effects of added soluble or aggregated Pin1 on the kinetics of Sup35 fiber formation will be determined. Pin1/Sup35 binding in vitro, and the relative cellular localization of [PSI+] and [PIN+] aggregates (using different color GFP fusions) will be examined. Other factors important for the de novo appearance of prions will be identified as genes that inhibit the induction of [PSI+] when mutated or overexpressed. Also, mutations will be isolated in the SUP35 prion domain that permit the induction of [PSI+] in the absence of [PIN+], and that stimulate the spontaneous appearance of [PSI+] in [PIN+] strains. The latter are analogous to "familial" mutations of the human PrP prion gene that cause a heritable predisposition to prion disease. We will also study: 1) stabilization of emerging prions by cloning a Mendelian mutation that prevents stabilization of an unstable [PSI+]; 2) maintenance and expression of existing prions by cloning high copy genes that cure or inhibit [PSI+], or are synthetic-lethal with [PSI+]; 3) effects of varying levels of chaperones or proteasome activity on induction or maintenance of [PSI+] and [PIN+]. The transmission of prions across species lines threatens human health. Using prions formed from heterologous Sup35, the hypothesis that different strains of prions can cross the species barrier with different efficiencies, will be tested. Strains of [PSI+], and mutations in SUP35, that alter the strength of the species barrier will be selected. The relative location of different species of prions in the same cell will be determined when the species barrier is crossed. The X-ray structure of Sup35 will be determined in a Sup35/Sup45 co-crystal. Sup45 should prevent the formation of Sup35 fibers that usually preclude crystal formation. Structural differences between Sup35 aggregates of different [PSI+] strains will be investigated using protease digestion and an assay based on effects of denaturation on immunoreactivity. "Pioneer" yeast prions will be identified (by 2D-gel analysis and mass spectroscopy) as proteins present in pellet fractions of yeast extracts, but soluble in lysates of isogenic strains cured of prions by both GuHCl and deletion of HSP 104. The identification of new prions will help elucidate the principles of how prions work and why they exist.